Cortisol Immunosensors: A Literature Review

Cortisol is a steroid hormone that is involved in a broad range of physiological processes in human/animal organisms. Cortisol levels in biological samples are a valuable biomarker, e.g., of stress and stress-related diseases; thus, cortisol determination in biological fluids, such as serum, saliva and urine, is of great clinical value. Although cortisol analysis can be performed with chromatography-based analytical techniques, such as liquid chromatography–tandem mass spectrometry (LC-MS/MS), conventional immunoassays (radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), etc.) are considered the “gold standard” analytical methodology for cortisol, due to their high sensitivity along with a series of practical advantages, such as low-cost instrumentation, an assay protocol that is fast and easy to perform, and high sample throughput. Especially in recent decades, research efforts have focused on the replacement of conventional immunoassays by cortisol immunosensors, which may offer further improvements in the field, such as real-time analysis at the point of care (e.g., continuous cortisol monitoring in sweat through wearable electrochemical sensors). In this review, most of the reported cortisol immunosensors, mainly electrochemical and also optical ones, are presented, focusing on their immunosensing/detection principles. Future prospects are also briefly discussed.


Introduction
The steroid hormone cortisol is the final product of the well-known hypothalamicpituitary-adrenal (HPA) axis, one of the four major neuroendocrine systems through which the hypothalamus and pituitary gland govern neuroendocrine function. Cortisol is synthesized in the zona fasciculata/reticularis cells of the adrenal cortex, under the control of adrenocorticotropic hormone (ACTH), and then secreted to the circulation. Approximately 80-90% of cortisol in blood plasma is bound to corticosteroid-binding protein (CBP), the rest is circulated bound to albumin, and only a small fraction, <10%, of total hormone is free circulating cortisol. Cortisol is lipophilic enough to cross the plasma membrane of target cells without the need of a membrane transporter. Within the target cell, cortisol binds to intracellular glucocorticoid receptors and the complexes formed translocate to the nucleus, where they may either enhance or decrease the expression of multiple genes. In this way, cortisol may affect many important biochemical functions, including release of ACTH, synthesis of gluconeogenesis-related enzymes, breakdown of muscle proteins, and lipolysis. Cortisol may be transformed into its inactive metabolite, unconjugated (free) forms. Measurement of 24 h UFC, which is not affected by diurnal variation, is considered to reliably reflect tissue exposure to free cortisol over a day, and has therefore been used as a well-established screening test for the diagnosis of Cushing's syndrome, as already mentioned [1,7,8]. Under normal conditions, urine cortisol levels have been reported to range between 21.5 and 149.7 µg/24 h [10] or between 10 and 100 µg/24 h [18].
Besides determination of cortisol levels in blood serum/plasma, saliva, or urine samples, measurement of cortisol extracted from human hair (usually, 1-3 cm of hair from the posterior vertex region of the scalp) has also been reported [19,20]. Since hair has been reported to grow at an average rate of 1 cm per month [21], hair samples allow determination of cumulative cortisol over a period of several months. In this regard, hair cortisol levels have been suggested as a retrospective, long-term biomarker of HPA axis functioning [20], stress-related, emotional and behavioral symptoms [22,23], or systemic exposure to cortisol in patients with Cushing's syndrome [1]. Hair cortisol levels have been reported to range between 18 and 153 pg/mg [10] and 1.7 and 153.2 pg/mg [18]. In addition, cortisol levels in fingernail samples may also serve as a putative retrospective biomarker [24,25]. Finally, measurement of cortisol metabolites in feces of animals has been used as a noninvasive means to evaluate release of glucocorticoids and thus adrenocortical activity in various animal species [26].
Cortisol immunoassays are based on antibodies recognizing cortisol and are the most frequently used methods for determining cortisol in routine clinical practice. Cortisol immunoassays include radioimmunoassays and ELISAs [12], as well as chemiluminescent immunoassays, electrochemiluminescence immunoassays, and fluorescence immunoassays [2,7]. In most cases, immunoassays are the current method of choice to determine total (bound and unbound) cortisol in serum/plasma as a fast and reproducible approach to estimate cortisol status; however, immunochemical determination of total cortisol in serum/plasma samples may be affected by changes in CBP and/or albumin levels [1,30]. Immunoassays are also the most frequently used method for determining urine free (unconjugated) cortisol (UFC) in clinical laboratories. Moreover, immunoassays have been widely applied to the determination of salivary cortisol, since they can provide the low detection limits required to quantitate the hormone present in the salivary samples, even at the nadir of the diurnal rhythm [1].
Despite their wide use, the analytical specificity of cortisol immunoassays may be limited by antibody cross-reactivity with other steroids present in the sample, while preanalytical interferences may also occur [15]. On the other hand, LC-MS/MS methods are characterized by high specificity and are progressively more and more often used in clinical laboratories [1], especially when interferences are suspected to occur [9]. Nevertheless, immunoassays are still preferred for cortisol determination and are considered the method of choice for assessing serum/plasma cortisol levels as a fast screening, especially in emergency cases.
Immunosensors are advanced analytical platforms, which-similarly to conventional immunoassays-are based on antibodies recognizing the analyte of interest [31]. Immunosensors exhibit a series of advantages in comparison with immunoassays, such as simple and fast assay protocols, which can be performed by nonexperts, as well as small detection modules, which allow their broader application, i.e., even outside the lab. Thus, the development of antibody-based sensors, especially at the point of care, and/or wearable sensors that would allow real-time cortisol monitoring in different body fluids, including sweat [32], has been receiving increasing attention as a powerful alternative to conventional immunoanalytical methods. Due to the continuously growing interest in the field, we considered it worthwhile presenting an updated review on reported cortisol immunosensors for the analysis of different biological samples.

Immunosensors for Detecting Cortisol in Biological Samples
Cortisol immunosensors were first reported in the mid-1990s. One of the first cortisol immunosensors was a noninvasive, reusable amperometric immunosensor, which was based on an anti-cortisol antibody for biorecognition and horseradish peroxidase (HRP) for signal generation. The antibody and the enzyme were co-immobilized on a chemically activated membrane, which had been mounted around the tip of an oxygen electrode. The assay principle was that the current created in the presence of the enzyme substrate was reduced upon binding of the antigen to the co-immobilized antibody [33]. At about the same time, the development of an invasive cortisol immunosensor was reported [34]. That sensor employed an HRP-labeled cortisol conjugate and an anti-cortisol antibody immobilized on the surface of a platinum electrode (working electrode). The platinum electrode was inserted into commercially available microdialysis probes and the probes were modified so as to bear the reference (Ag/Cl) and counter (Ag) electrodes on their top and then implanted in the jugular vein and used for real-time determination of cortisol in conscious animals, by monitoring peroxidase activity [34]. Since then, many cortisol immunosensors-mostly noninvasive-have been reported in the literature. Cortisol immunosensors reported thus far are mainly electrochemical and optical, while one of the first immunosensors described for cortisol was piezoelectric [35].
Two previous review articles have provided accumulated information on immunosensors, both electrochemical and optical ones, for determining cortisol-usually among other health biomarkers-focusing on the analysis of saliva samples [28,36]. An informative table summarizing reports on various cortisol sensors based on different detection techniques was included in a recent article [37]. However, the information currently available in the literature might be considered, at least in our opinion, somewhat "fragmented."

Electrochemical Cortisol Immunosensors
The first cortisol immunosensors, reported in the mid-1990s [33,34] as already mentioned, were electrochemical. In the meantime, many more electrochemical immunosensors for cortisol have been described in the literature, while some recent review articles have provided accumulative information on cortisol electrochemical sensors [10,38].
During the last decade, various wearable electrochemical sensing platforms, which in general are considered ideal for analyzing sweat samples [39], have been developed and applied to the immunodetection of cortisol in sweat, as critically presented in previous review papers [18,40]. Several electrochemical cortisol immunosensors for sweat analysis have been described in the literature during the last couple of years (2020-2022) [41][42][43][44][45][46]. Tear analysis by means of electrochemical cortisol immunosensing has also been reported [47].

Resistance
Human saliva and buffer solution of rat adrenal gland acute slices 10 pg/mL [58] Noncompetitive (Direct binding of cortisol to an anti-cortisol Ab immobilized on Au-substrates modified with ZnO nanostructures (1D nanorods, 2D nanoflakes)) Cyclic voltammetry Saliva 1 pM [59] Noncompetitive (Direct binding of cortisol to an anti-cortisol Ab covalently immobilized on microfabricated interdigitated microelectrodes) Cyclic voltammetry Saliva 10 pg/mL-100 ng/mL/ 10 pg/mL [60] Competitive (Competition between a GOD 3 -cortisol conjugate and free cortisol for binding to an anti-cortisol Ab immobilized on platinum electrodes; lateral and vertical fluid control mechanisms were integrated in the sensor) Amperometry Saliva 0.1-10 ng/mL [61] Noncompetitive (Direct binding of cortisol to an anti-cortisol Ab immobilized on gold microelectrode arrays) Electrochemical impedance spectroscopy Saliva and interstitial fluid 1 pM-100 nM [63] Competitive (Competition between free cortisol and a cortisol analog covalently immobilized on single-walled carbon nanotubes and free cortisol for binding to an anti-cortisol Ab) Resistance/conductance Saliva 1 pg/mL-10 ng/mL/ 1 pg/mL [62] Competitive (Competition between cortisol immobilized on naflon pretreated glassy carbon electrodes and free cortisol for binding to a biotinylated anti-cortisol Ab; detection was performed via reaction with HRP-streptavidin)  Competitive (Competition between an ALP-labeled cortisol conjugate and free cortisol for binding to an anti-cortisol Ab immobilized through protein A on magnetic particles; the immunocomplexes formed were trapped on the surface of screen-printed electrodes with a small magnet and ALP activity was monitored) Differential pulse voltammetry Serum 5 × 10 −3 -150 ng/mL/ 3.5 pg/mL [69] Competitive (Competition between an HRP-labeled cortisol conjugate and free cortisol for binding to an anti-cortisol Ab immobilized on gold electrodes functionalized with a AuNP-protein G-DTBP 4  Among the most recently reported electrochemical cortisol immunosensors are the following (Table 1): (a) an electrochemical immunosensor for sweat cortisol based on an L-cys/-gold nanoparticles/-MXene(titanium carbide)-modified thread electrode [41]; (b) a photoelectrochemical immunosensor for salivary cortisol based on the competition between free cortisol and a bovine serum albumin (BSA)-cortisol conjugate immobilized onto magnetic beads for binding to an anti-cortisol antibody labeled with silver nanoclusters (AgNCs); after formation of the immunocomplexes, the magnetic beads were separated, the AgNCs were dissolved in nitric acid and the silver ions produced were transferred to the sensor electrode for ion exchange with cadmium sulfide (CdS) quantum dots, which led to a decrease in the photocurrent intensity [50]; (c) a battery-free, wireless and flexible electrochemical patch-type immunosensor for sweat cortisol determination employing near-field communication with a smartphone (Figure 1); the sensor employed an anti-cortisol antibody covalently immobilized on screen-printed electrodes, coated with gold nanoparticles (AuNPs) through a bifunctional polyethylene glycol (PEG) derivative [42]; (d) an electro-Biosensors 2023, 13, 285 7 of 17 chemical immunosensor for sweat cortisol based on a flexible electrode that was developed by transferring a multiwalled carbon nanotube (MWCNT) film on a polydimethylsiloxane substrate and subsequently depositing gold nanoparticles on the MWCNT surface [43]; (e) an electrochemical impedance spectroscopy immunosensor for detecting cortisol as a stress marker in saliva of trainee guide dogs during the training process [37]; (f) an immunosensor for salivary cortisol based on a gold nanoparticle-molybdenum disulfide-gold nanoparticles scaffold as transducer combined with a smartphone-operated point-of-care miniaturized differential pulse voltammetry system [52]; (g) an immunosensor employing a modified tin-doped indium oxide (ITO) electrode, on which an anti-cortisol antibody labeled with ferrocene tags had been immobilized; a point-of-care electrochemical platform was thus constructed and applied to determining cortisol in artificial saliva and whole-body zebrafish with or without extraction [51]; (h) an immunosensor using an integrated wireless sensing device that was based on laser-engraved graphene electrodes for detecting sweat cortisol; the cortisol diurnal cycle in human sweat was investigated with that immunosensor and a strong empirical correlation between serum and sweat cortisol was reported [44]; (i) a dual amperometric immunosensor microchip for the simultaneous detection of cortisol and insulin in untreated serum samples; insulin detection was based on a peroxidase-labeled sandwich assay, and cortisol detection on an alkaline phosphatase-labeled (ALP-labeled) competitive immunoassay [64]. Most of the electrochemical cortisol immunosensors are characterized by very good analytical features, such as high sensitivity, with LOD values in the range of pg/mL or even fg/mL, as shown in Table 1. Immunosensor specificity is mainly dependent on the anti-cortisol antibody employed, while other sensor reagents/components along with the biological matrix of the sample analyzed may also affect specificity, at least to some extent. For several electrochemical immunosensors [41,53,54,57], high reproducibility has been reported. However, larger validation studies are required before further sensor exploitation, as we mention in Section 3.

Optical Cortisol Immunosensors
The first optical cortisol immunosensors were developed in the late 2000s (Table 2). These sensors, based on the surface plasma resonance (SPR) detection principle [77][78][79] and applied to the detection of salivary cortisol, are mentioned in a recent review article concerning analysis of saliva as an ideal "health mirror" sample [80]. Since the late 2000s, several other optical cortisol immunosensors have been described in the literature.
In this work, we present most of the articles published in the last 15 years regarding the development of optical immunosensors for detecting cortisol in a variety of matrices, from plain buffer solutions [81-83] to complex biological specimens. As shown in Table 2, complex biological samples analyzed for cortisol with optical immunosensors include mainly saliva [78,79,84-90], as well as blood plasma/serum [91,92] and urine [78]. Most of the electrochemical cortisol immunosensors are characterized by very good analytical features, such as high sensitivity, with LOD values in the range of pg/mL or even fg/mL, as shown in Table 1. Immunosensor specificity is mainly dependent on the anti-cortisol antibody employed, while other sensor reagents/components along with the biological matrix of the sample analyzed may also affect specificity, at least to some extent. For several electrochemical immunosensors [41,53,54,57], high reproducibility has been reported. However, larger validation studies are required before further sensor exploitation, as we mention in Section 3.

Optical Cortisol Immunosensors
The first optical cortisol immunosensors were developed in the late 2000s (Table 2). These sensors, based on the surface plasma resonance (SPR) detection principle [77][78][79] and applied to the detection of salivary cortisol, are mentioned in a recent review article concerning analysis of saliva as an ideal "health mirror" sample [80]. Since the late 2000s, several other optical cortisol immunosensors have been described in the literature. In this work, we present most of the articles published in the last 15 years regarding the development of optical immunosensors for detecting cortisol in a variety of matrices, from plain buffer solutions [81][82][83] to complex biological specimens. As shown in Table 2, complex biological samples analyzed for cortisol with optical immunosensors include mainly saliva [78,79,[84][85][86][87][88][89][90], as well as blood plasma/serum [91,92] and urine [78].
Among the most recently reported (2020-2022) optical cortisol immunosensors are the following ( Table 2): (a) an optical immunosensor for real-time/continuous monitoring of cortisol in human blood plasma obtained after filtration or through microdialysis employing a complex immunoassay setup for cortisol biosensing through particle mobility monitoring with the aid of a microscope [91]; (b) an SPR immunosensor based on D-shaped optical fibers; the sensor has been applied to determining cortisol in buffer solutions [81]; (c) an SPR immunosensor based on an unclad plastic optical fiber, first coated with gold/palladium alloy and subsequently loaded with an anti-cortisol antibody; this sensor has also been applied to determining cortisol in buffer solutions [82]; (d) an optical immunosensor based on metal (gold)-enhanced time-resolved fluorescence for the continuous real-time monitoring of cortisol in buffer solutions [83]; (e) a paper-based optical immunosensor for serum cortisol, based on a competitive assay principle and employing an anti-cortisol antibody labeled with gold nanoparticles (Figure 2) as signal indicator [92]; (f) an optical immunosensor for salivary cortisol based on fluorescence quenching caused by cortisol binding to quantum dots loaded with an anti-cortisol antibody (or with an aptamer) [84]. Although optical cortisol immunosensors may be considered less sensitive than electrochemical ones, at least on average, most of them are characterized by adequate sensitivity, with LOD values mainly in the range of ng/mL, as shown in Table 2. Integration of Figure 2. Schematic representation of: (A) production of paper-based sensors using a wax printer; (B, i) immobilization of the BSA-cortisol conjugate on the detection surface; (B, ii) competitive assay between cortisol in the sample and BSA-cortisol immobilized on the detection surface of the paper sensor for binding to an anti-cortisol antibody labeled with gold nanoparticles; (C) collection and interpretation of the results using image capture/processing programs. (Adopted with permission from [92]).
Although optical cortisol immunosensors may be considered less sensitive than electrochemical ones, at least on average, most of them are characterized by adequate sensitivity, with LOD values mainly in the range of ng/mL, as shown in Table 2. Integration of advanced nanomaterials, especially during the last few decades, has led to signal enhancement and contributed to achieving increased analytical sensitivity. Similarly to electrochemical immunosensors, specificity of the optical cortisol immunosensors is mainly dependent on the specificity of the primary antibody employed, while it might also be affected by other reagents/components used for constructing the immunosensing platform and/or matrix of the biological samples. For some of the optical immunosensors [81], high reproducibility has been reported. Nevertheless, as in the case of electrochemical sensors, large validation studies are required before reaching a solid evaluation.

Discussion-Future Perspectives
Cortisol homeostasis is essential for human health, and abnormal cortisol levels have been correlated with and may serve as a valuable biomarker for several disease states. Thus, it is important to monitor cortisol concentrations in various biological samples by means of proper analytical methods. Cortisol immunoassays are currently considered the analytical method of choice for determining cortisol in biological specimens, such as blood serum/plasma, urine, saliva, or, more recently, hair. Transformation of the conventional immunoassays to technologically advanced antibody-based assays, which can easily be accomplished in a short time by unskilled persons and are capable of being "integrated" into portable devices for point-of-care measurements, has led to the development of several cortisol immunosensors during the last three decades. Cortisol immunosensors can be divided according to the signal transduction principle they rely upon. Most cortisol immunosensors are electrochemical and rely on signal measurement through cyclic voltammetry, impedance spectrometry, and amperometry. On the other hand, several optical immunosensors, including flow lateral-type strip sensors, have been developed, especially during the last decade. Besides the usually measured optical signals, e.g., SPR signals, some of the most recently reported cortisol immunosensors [91] rely on the measurement of other parameters, e.g., on particle mobility monitoring with the aid of a microscope [112].
Some immunosensors can simultaneously detect cortisol along with another biomarker, e.g., insulin [64], lactate [56] or IL-6 [75]. This "multianalyte" approach, although technologically difficult to achieve and therefore rarely reported, is highly desirable from a clinical point of view. Thus, a dual electrochemical immunosensor proposed for the simultaneous detection of cortisol and insulin at the point of care [64] may eventually offer improved management of diabetes.
A great number of cortisol immunosensors have been applied to the analysis of saliva samples and less to urine or blood plasma/serum, while too little information concerning immunosensors for hair cortisol is currently available, at least to our knowledge. This tendency might be attributed, at least partly, to special requirements for the collection and/or treatment of the relevant samples, e.g., the requirement of 24 h urine collection renders urine samples weak candidates for real-time detection of cortisol through a point-of-care immunosensor device [40]. Other factors supporting this trend may include matrix complexity, the need for careful pH adjustment before sample analysis, etc. On the other hand, a special group of electrochemical immunosensors (e.g., miniaturized and flexible/wearable sensors that are based on new materials, such as two-dimensional nanosheets of MoS 2 [74]) have allowed real-time and even continuous monitoring of cortisol in sweat. As reported, free (protein-unbound) cortisol seems to be present in sweat glands, through a mechanism resembling transportation of free cortisol by the bloodstream to the salivary glands. From sweat glands, cortisol is thought to reach sweat by passive transportation through the cell lipid bilayer membrane [113]. Sweat cortisol concentrations have been reported to range from 8 to 142 ng/mL [10,18], the highest levels being found in the morning and correlating with salivary levels [113]. So far, sweat cortisol has not been detected with conventional methods-at least, not routinely-possibly due to difficulties in collecting and properly storing the corresponding samples for subsequent laboratory analysis.
At present, routine analysis of biological samples for cortisol monitoring is only performed in lab settings [114]. Further research is needed before cortisol immunosensors have become fully commercialized and available to clinical and self-monitoring applications. A first challenge of such efforts would be to perform large validation studies so as to ensure that the analytical characteristics of the immunosensors developed are of high quality. Although there is always space for improvement, sensitivity/LOD is usually not a problem, while specificity and/or simplicity of production and cost-related issues associated with the anti-cortisol antibodies, which are inherent in all immunochemical analytical techniques, might be addressed by antibodies' replacement with aptamers or MIPs. However, special attention should be paid to the validation of repeatability/reproducibility [115] as well as operational stability, especially when complex biological samples, such as blood plasma/serum are to be analyzed (which may affect integrity and deteriorate functioning of electrodes in electrochemical immunosensors). Conditions of reusability as well as storage stability/durability/life span are also issues to be studied. Wearable immunosensors for detection of cortisol, mainly in sweat samples, seem to provide exciting prospects for further progress in the field, but particular aspects have to be addressed in the years to come [116]. These aspects include accurate sample collection, potential toxicity and biocompatibility of sensor materials, and appropriate power supply of the flexible electronics these sensors require, while data processing and communication constitute a separate research field, which may be further elaborated and improved. Last, but not least, detailed and thorough knowledge of the biological and chemical characteristics of the samples to be analyzed along with deep insight in cortisol physiology and partitioning/kinetics/dynamics of the hormone in different compartments/fluids of the organism in normal and disease states would nicely supplement research in the field (Figure 3), and relevant studies may be performed in parallel. To achieve this, close collaboration among clinicians, physicists, chemists, bioscientists, and engineers is a prerequisite. a separate research field, which may be further elaborated and improved. Last, but not least, detailed and thorough knowledge of the biological and chemical characteristics of the samples to be analyzed along with deep insight in cortisol physiology and partitioning/kinetics/dynamics of the hormone in different compartments/fluids of the organism in normal and disease states would nicely supplement research in the field (Figure 3), and relevant studies may be performed in parallel. To achieve this, close collaboration among clinicians, physicists, chemists, bioscientists, and engineers is a prerequisite. Figure 3. Cortisol has been detected in various biological fluids, and cortisol levels may serve as a valuable biomarker (e.g., of stress). Cortisol determination has been achieved mainly with analytical methods based on specific cortisol binders and especially anti-cortisol antibodies. Cortisol immunosensors based on different signal transduction principles are expected to be eventually commercialized and serve as an easy-to-handle, reliable tool for point-of-care clinical analysis of cortisol.

Conclusions
Most reported cortisol immunosensors are currently at the proof-of-concept stage, and further research is necessary before the most appropriate among them could eventually become commercially available. Provided that all issues requiring further elucidation and thorough validation, as discussed in the present work, can be resolved, cortisol im- Figure 3. Cortisol has been detected in various biological fluids, and cortisol levels may serve as a valuable biomarker (e.g., of stress). Cortisol determination has been achieved mainly with analytical methods based on specific cortisol binders and especially anti-cortisol antibodies. Cortisol immunosensors based on different signal transduction principles are expected to be eventually commercialized and serve as an easy-to-handle, reliable tool for point-of-care clinical analysis of cortisol.

Conclusions
Most reported cortisol immunosensors are currently at the proof-of-concept stage, and further research is necessary before the most appropriate among them could eventually become commercially available. Provided that all issues requiring further elucidation and thorough validation, as discussed in the present work, can be resolved, cortisol immunosensors will be an invaluable analytical tool that will enrich and expand the capabilities of the existing methodology, thus offering exceptional prospects in the field of clinical analysis of cortisol.